Use of erythropoietin for the preventive or curative treatment of cardiac failure

ABSTRACT

Provided are uses of erythropoietin, or a derivative or functional analogue thereof, for the production of a medicament for the preventive or curative treatment of patients suffering from, or at risk of suffering from, cardiac failure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/NL03/00011, filed Jan. 9, 2003,published in English as International Patent Publication WO 03/057242 onJul. 17, 2003, which claims the benefit under 35 U.S.C. § 119 ofInternational Patent Application PCT/NL02/00010, filed Jan. 9, 2002.

TECHNICAL FIELD

The invention relates to the field of medicine. More particularly, thepresent invention relates to the treatment of hypoxia-related disordersin mammals and compounds and pharmaceutical preparations for usetherein.

BACKGROUND

Cardiac failure is a chronic clinical syndrome characterized by theheart being unable to adequately pump blood throughout the body.Generally, it is caused by any disease or condition that causes loss ofcardiac tissue, especially of the left ventricle. The most common causesinclude cardiac infarction, coronary artery disease, myocarditis,chemotherapy, alcoholism and cardiomyopathy. On the other hand, cardiacfailure may be caused by diseases or conditions that require anexcessive demand for cardiac output. The most common causes includehypertension, valvular heart diseases (most often mitral insufficiencyand aortic stenosis) and disorders of the thyroid gland. The long-termextra demand on the heart will lead to a compensatory hypertrophy of thecardiomyocytes. As the capillary network does not extend, hypertrophywill lead to a relative ischemia because the diffusion pathway foroxygen will increase. Recently, the importance of the role of ischemiain cardiac failure has been put forward (Van den Heuvel et al., 2000).

Thus far, the treatment of patients suffering from ischemic heartdisease and subsequent cardiac damage leading to heart failure hasfocused on early reperfusion. Although additional cell protectiontherapy might, in theory, limit the damage that is caused by myocardialischemia and hence, reduce morbidity and mortality, no sufficienttherapies exist to date.

Additional supportive therapy to protect the myocardium in acuteischemic conditions consists nowadays in administration ofbeta-blockers, calcium antagonists and nitrates. However, thesetherapies have a low efficacy and alternative and/or additionalstrategies are needed.

SUMMARY OF THE INVENTION

The present invention provides for the use of erythropoietin (EPO), orderivatives or functional analogues thereof, for the preparation of amedicament for the preventive and/or curative treatment of patientssuffering from, or at risk of suffering from, cardiac failure. Treatmentwith EPO for these conditions can be beneficial, irrespective of theircause and nature. The invention also provides a method for treating apatient suffering from, or at risk of suffering from, cardiac failure,the method comprising a step of administering to the patienterythropoietin, or a derivative or functional analogue thereof. In oneaspect of the invention, the patient suffering from heart failure is notanemic. Although recent clinical studies demonstrated the beneficialeffects of EPO in patients with congestive heart failure (CHF) that alsohad anemia (Silverberg et al., 2000 and 2001), the person skilled in theart before the present invention would not treat patients with heartfailure by using EPO in the absence of specific other indications forthe use of EPO, such as anemia, kidney disease or leukemia. A certainfraction of CHF patients is anemic (low hematocrit/low hemoglobinpercentage) and a correlation exists between the severity of thecondition of CHF and the degree of anemia. When patients with anemia inCHF were treated with recombinant EPO, an improvement with respect tocardiac function, renal function and a decrease in the need fordiuretics and hospitalization was observed (Silverberg et al. 2000 and2001). Other publications (EP0813877; Mancini et al., 2001) alsodescribe the use of EPO to raise the red blood cells and/or preventanemia in the case of congestive heart failure. It appears that thusfar, the improved condition of heart patients, upon treatment with EPO,was ascribed to the purposeful hematocrit elevation when patients had amedical indication to treat them with EPO, thus improving peripheraloxygenation by a mechanism unrelated to a change in cardiac function.The present invention for the first time discloses the use of EPO forthe treatment of heart failure irrespective of whether the hematocritvalue (red blood cell count) of the patient is lower than normal or not.This provides cardiac failure per se as a novel indication for the useof EPO. The present invention therefore provides for the use of EPO fortreatment of patients with heart failure, wherein the patients do notnecessarily have another indication besides heart failure, which wouldotherwise have warranted the treatment of such a patient with EPO basedon the presently available knowledge.

In certain embodiments, the EPO, or derivative or functional analoguethereof, has been produced in a host cell expressing at least the E1Aprotein of an adenovirus, preferably in a host cell derived from aPER.C6™ cell.

The invention further provides erythropoietin, or a functional part,derivative and/or analogue thereof, for treatment of a patient sufferingfrom, or at risk of suffering from, a chronic and/or acute coronarysyndrome. Preferably, EPO has been recombinantly produced on a host cellthat expresses at least the E1A protein of an adenovirus, morepreferably on a host cell derived from a PER.C6™ cell. Although the useof EPO to protect the myocardium from acute ischemic injury has beendescribed (see WO 00/61164, WO 01/82952), the EPO used may cause aconcomitant significant increase in hematocrit values, which can beregarded as an undesired side effect for this application. The use ofEPO derived from PER.C6™ or another E1A-expressing host cell, leads toless of this side effect and, therefore, is beneficial (see alsoPCT/NL02/00686 for the demonstration that EPO produced on PER.C6™ isfunctional but gives rise to less increase in hematocrit values whencompared with a commercially available EPO preparation (EPREX®)).

The invention further provides the use of erythropoietin, or derivativesor functional analogues thereof, for the preparation of a medicament forthe preventive and/or curative treatment of chronic and/or acutecoronary syndromes. The invention also provides pharmaceuticallyeffective preparations comprising EPO or a derivative or functionalanalogue thereof for such treatments.

Furthermore, the invention provides methods for treating a patientsuffering from, or at risk of suffering from, undesirable effects ofchronic or acute coronary syndromes, comprising the steps ofadministering to the patient erythropoietin or a derivative or analoguethereof in an amount sufficient to prevent or reduce the undesirableeffects. Undesirable effects that may be decreased and/or inhibited bythe compounds of the present invention include detrimental effects, suchas apoptosis and/or necrosis of heart muscle cells. The effects on suchcells most likely occur through the interaction of compounds of theinvention with receptors present on such cells. Direct effects broughtabout by compounds of the present invention also include angiogeniceffects through which certain hypoxia-related coronary syndromes arereduced in severity, both in acute as well as in chronic cases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Real Time RT-PCR of EPO-R mRNA. Specificity was checked with theuse of restriction enzyme (NciI) for partial digestion of the 72 bpEPO-R product in expected fragments (39 bp and 34 bp).

FIG. 2. Western blot. Lane 1-3: MAPK (pERK1=44 kD; pERK2=42 Kd) in shamtreated hearts; lane 4-6: MAPK in EPO treated hearts; lane 7: EPO insham treated heart; lane 8: EPO-R in sham treated heart.

DETAILED DESCRIPTION

Erythropoietin (EPO), EPO derivatives and functional analogues are, whenappropriate, hereinafter referred to as “EPO” for the sake of brevity.EPO is a protein well known for its role in differentiatinghematopoietic stem cells into red blood cells, but it has manyadditional functions as well. This application reveals a novel EPO andEPO-receptor (EPO-R) system in the heart, which knowledge, according tothe present invention, is converted into practical use by administeringEPO to patients with heart failure.

Cardiac failure, also called heart failure, or chronic heart failure orcongestive heart failure, is defined as a heart disease in which theheart is not able to pump blood at a rate required by the metabolizingtissues, or when the heart can do so only with an elevated fillingpressure. Treatment of heart failure with EPO, according to theinvention, includes treatment of patients having or being at risk ofhaving cardiac infarction, coronary artery disease, myocarditis,chemotherapy, alcoholism, cardiomyopathy, hypertension, valvular heartdiseases (most often mitral insufficiency and aortic stenosis) anddisorders of the thyroid gland and the like.

According to the invention, a patient can be human, but may also includean animal with heart failure. Therefore, treatment according to theinvention may pertain to humans as well as to other animal species.

A “non-anemic patient” as used herein, is a patient that has ahemoglobin value that is considered as being within the normal range,which value would not lead a physician to prescribe EPO to this patient.Until now, application of EPO has been restricted to the prevention orcorrection of anemia in specific patient populations, including the(pre)dialysis phase of chronic renal insufficiency, cytostatic therapy,premature infants and as preparation for autologous blood transfusion orsurgical procedures with anticipated major blood loss. The general aimin such cases is to increase hemoglobin levels (Hb) by increasing thenumber of red blood cells (hematocrit) to a specific range by adaptingstandard dosage regimes to individual needs. Depending on the patientpopulation, the optimal Hb level ranges from a lower limit of 6.5-7.5mmol/L to an upper limit of 8.0-8.7 mmol/L.

According to one aspect of the invention, the EPO administered orformulated for use in the treatment of myocardial disease is EPO as maybe isolated from any suitable source. Preferably, human EPO isrecombinantly produced and isolated from a suitable recombinant hostcell and/or from the culture medium. In the case of recombinantproduction, the host may suitably be chosen from any cell capable ofrecombinantly producing protein, such as bacterial host cells (e.g., E.coli, B. subtilis), yeast (e.g., S. cerevisiae, K. lactis), fungi (e.g.,A. niger, Pichia), and mammalian cells (e.g., CHO, BHK cells) includinghuman cells. According to one aspect of the invention, EPO isrecombinantly produced in an immortalized human cell line, in particularPER.C6™ (ECACC deposit nr. 96022940). It is also possible to administerEPO in a gene-therapy setting according to the invention, for instance,by treating a patient with a vector comprising a nucleic acid sequencecapable of expressing EPO when delivered to a target cell.

Derivatives of EPO refer to modifications of the source EPO, which maybe urinary EPO or EPO recombinantly producible from a cDNA or genesequence, wherein the expression product has one or more modificationsrelative to the source EPO, which modifications may be in the primarystructure by substitution of one or more amino acid residues (such as inNESP), deletion, addition or relocation of one or more amino acidresidues, or alterations in the post- or peri-translational modificationof the protein backbone, such as hydroxylations, phosphorylations orglycosylations of amino acid residues, sulphur bridges, and the like.

Derivatives also encompass naturally or non-naturally occurring EPOvariants coupled to non-EPO-related proteinaceous moieties or even tonon-proteinaceous moieties. Derivatives of EPO are encompassed by theinstant invention, as long as they interact with the EPO receptor andcause a reduction or prevention of the undesirable effects caused bychronic or acute coronary syndromes that include, but are not limitedto, myocardial ischemia, myocardial infarction or heart failure, orcaused by hypoxia conditions in the heart in general. As a measure forthe occurrence of undesirable effects, the degree of apoptosis and/ornecrosis in the heart tissue and/or the levels of purines in thecoronary effluent circulation may be determined, or by any other meansknown in the art.

Functional analogues of EPO refer to molecules not necessarily derivedfrom naturally on non-naturally occurring EPO that are capable ofmimicking the interaction of EPO with its receptor, whereby theundesirable effects caused by chronic or acute myocardial ischemia ormyocardial infarction, or hypoxia in the heart in general, are reducedand/or prevented. Such functional analogues may comprise peptidomimeticsand/or non-peptidic molecules mimicking the idiotope interacting withthe EPO-R. It will be understood by those of skill in the art that thefunctional analogue according to the invention need not necessarilyinteract with the same idiotope or in the same way, as long as it mimicsthe interaction of EPO with its receptor. Functional analogues maysuitably be screened and selected from (synthetic) peptide libraries,phage or ribosome polypeptide display libraries, or small moleculelibraries. Those of skill in the art are capable of screening for ordesigning functional analogues and test their functionality in assaysdisclosed herein. In addition to assays based on apoptosis and/or purinedetermination, other methods, such as methods towards measuring cellnecrosis that are generally known in the art, may be used to test thefunctionality of the analogue in reducing and/or preventing theundesirable effects of hypoxia.

EPO may be administered to a mammal in any pharmaceutically acceptableform. Generally, EPO will be administered parenterally or subcutaneously(sc), but the way of administration may vary from time to time. Wheneverit is needed to obtain a quick response, it may be desirable to add EPOin high dose form by means known to quickly deliver the pharmaceuticalto the heart. Instances where this is clearly desired are, for example,where the patient suffers from acute syndromes such as acute myocardialischemia, myocardial infarction or acute heart failure. In thesecircumstances, doses typically rise above the doses that areadministered to human patients suffering from anemia or suffering fromchronic coronary syndromes (Silverberg et al. 2000 and 2001). Normaldoses that are administered to adult renal failure patients are in therange of 4000-7500 IU per week (80-100 kg body weight). These amountsare normally divided into three separate doses per week fan thecommercially available epoetin alpha or EPREX® (EPO produced on CHOcells). Higher doses for the treatment of acute coronary disorders maybe given daily or even more frequently. The maximum tolerable dose mayhave to be determined in order to prevent hematocrit values andhemoglobin concentrations to rise too sharply. Persons of ordinary skillknow how to monitor hematocrit values and hemoglobin concentrations inpatients to prevent undesired side effects, such as extreme high bloodpressure that may occur in later stages of the treatment. Theseadministration schemes contrast the schemes used by Silverberg et al.(2000 and 2001) to treat anemic patients that suffer from congestiveheart failure, where administration of EPO was prolonged for weeks oreven months. For acute coronary syndromes, it might not be necessary toprolong the treatment with the high doses for several months, since theprotective effect is required instantly and undesired side effects mightoccur when such high doses are given for prolonged periods of time. Inthe case of chronic coronary syndromes including, but not limited to,myocardial ischemia or heart failure, lower doses may be administeredduring a longer time interval. Heart failure includes both acute heartfailure syndromes, such as in the frame of myocardial infarction, butalso reduced pumping of the heart in chronic cases. These applied dosesare comparable to doses given to renal failure patients that suffer fromthe lack of EPO. Doses for non-acute hypoxia-related myocardialdisorders may range from 10 to 10,000 IU per administration, preferably,1000 to 2500 IU per administration (for an adult of 80-100 kg). Also, inthis case, monitoring may be necessary to prevent unwanted side effects.

As disclosed in WO 00/63403, EPO can also be recombinantly produced onPER.C6™ cells. It was recently described (see patent applicationPCT/NL02/00686) that EPO thus produced leads to a significantly lowerincrease of the hematocrit value upon administration than similar dosesof recombinant EPO currently commercially available (EPREX®). Thisappears mainly due to the specific posttranslational modifications ofthe EPO thus produced, which appear related to the presence of at leastthe E1A sequence of an adenovirus in expressible format in the host cellused for recombinant production of EPO. A less pronounced increase inhematocrit value upon administration of EPO is beneficial for useaccording to the present invention. It is, therefore, a preferredembodiment of the present invention to use EPO according to theinvention, whereby the EPO has been recombinantly produced in a hostcell expressing at least the E1A protein, or a derivative or functionalanalogue thereof (see PCT/NL02/00686). Preferably, the host cell is aPER.C6™ cell. Such EPO can be used according to the invention for bothchronic and acute coronary syndromes.

Novel formulations of EPO-like proteins are known in the art. The NovelErythropoiesis Stimulating Protein (NESP) is known to be effective forlonger periods of time due to its modified glycosylation pattern, whichmakes the administration schedule such that only a once a week dose isrequired to sort the effects that were formerly found with three doses aweek of the original recombinant EPO protein. For the treatment of acuteor chronic coronary syndromes, it might also be useful to apply NESP,which should be administered in a similar way as described above forEPO, namely, at higher (and possibly more frequent) doses in the case ofacute coronary syndromes and at comparable (and equally frequent) dosesin the case of chronic heart failure. It remains to be seen whether themodified glycosylation of NESP as compared to EPO has anydifferentiating effect on the EPO-R present on myocytes and endothelialcells in the blood vessels of the heart.

Pharmaceutically acceptable formulations according to the inventiontypically comprise EPO according to the invention, usually together withpharmaceutically acceptable excipients, diluents, solvents, andoptionally, compounds acting in an additive or even synergistic fashion.Compounds of the latter category comprise compounds of the statinfamily, such as lovastatin, simvastatin, angiotensin-converting enzymeinhibitors (ACE-inhibitors), and the like.

It is worth noting that, according to the invention, the protectiveeffect of EPO on hypoxia-induced myocardial damage, as determined bypurine analysis in the coronary effluent and/or the degree of apoptoticcells in the myocardium, is observed within minutes after subcutaneousadministration. It is difficult to imagine that this effect should beascribed to EPO's known stimulating effect on angiogenesis, or to itshematopoietic effect for that matter, since these effects are typicallynot observed within the time frame of minutes, but rather days or evenweeks. It is tempting, therefore, to speculate that the cell protectiveeffect of EPO observed within minutes after administration is broughtabout by a direct intervention of EPO and tissues of, or in directcontact with, the myocardium. The fact that the EPO-R is found to beexpressed on the cell surface of the myocytes (as is shown in thisinvention), strongly suggests that direct anti-apoptotic andanti-necrotic effects occur through the action of EPO on thesereceptors, while the direct angiogenic effects of EPO most likely occurthrough the EPO-R expressed on endothelial cells in the capillaries.This effect may occur in vitro as well as in vivo.

The invention will now be illustrated by the following examples.

EXAMPLES Example 1 Detection of EPO and EPO-R in Normal Human and RatHeart Tissue

It has been found that EPO and the EEO-R are expressed in fetal cardiactissue (Juul et al. 1998). Despite the increasing body of literature onthe expression of EPO and its receptor and the putative roles associatedtherewith, little, if anything, is known of the distribution of EPO andEPO-R in adult heart tissue.

Expression of EPO and EPO-R was examined by real-time RT-PCR, westernblotting and immunohistochemistry on rat heart tissue and by westernblotting and immunohistochemistry on human heart biopsies.

Rat Heart (Langendorff Set-Up)

For this, ischemic/reperfusion (I/R) experiments in isolated rat heartssuspended in a so-called Langendorff apparatus (Van Gilst et al. 1988)were performed with and without the administration of EPO, using methodsgenerally known to persons skilled in the art.

Male Sprague Dawley Rats weighing approximately 300 grams (n=12) weredivided into four experimental groups. Two groups received globalcardiac ischemia by reducing coronary flow to 0.6 ml/minute for 30minutes followed by reperfusion for 45 minutes. Two other groups werewithout ischemia. Within each of the groups, half of the rats weretreated with EPO (10 U/ml) and half with saline. Rats were anesthetizedand 500 U of heparin was injected in the tail vein. The heart wasrapidly excised and the aorta was immediately retrogradely perfused by amodified Tyrode solution (glucose 10, NaCl 128.3, KCl 4.7, NaHCO₃ 20.2,CaCl₂, 1.35, NaH₂PO₄0.42, MgCl₂, 1.05; all mmol/liter) and wasequilibrated with 95% O₂ and 5% CO₂. Perfusion pressure was maintainedat 60 mmHg. Coronary flow (CF) was measured by a microprocessor, whichcontrolled the perfusion pressure by adjusting the peristaltic perfusionpump. CF, heart rate (HR), and left ventricular peak pressure weremonitored continuously. After equilibrating for five minutes, heartswere perfused for 20 minutes with EPO or saline before the I/R protocolstarted.

Real-Time RT-PCR

Total RNA was isolated from rat left ventricle and processed asdescribed previously (Brundel et al., 1999). Briefly, cDNA wassynthesized by incubating 1 μg of RNA in reverse transcription buffer,200 ng of random hexamers with 200 U of Moloney Murine Leukemia VirusReverse Transcriptase, 1 mmol/L of each dNTP, and 1 U of RNase inhibitor(Promega). Synthesis reaction was performed for 10 minutes at 20° C., 20minutes at 42° C., 5 minutes at 99° C., and 5 minutes at 4° C. Allproducts were checked for contaminating DNA. Fragments of EPO-R wereamplified (Forward primer: CAGGACACCTACCTGGTATTGGA (SEQ ID NO:1);reverse primer: CAGGCCCAGAGAGGTTCTCA (SEQ ID NO:2), Eurogentec, Belgium)with a GeneAmp® 5700 (Perkin-Elmer/ABI) employing a 40 cycle protocolconsisting of 30 seconds at 94° C., 1 minute at 56° C. and 30 seconds at72° C. After the last cycle, the 72° C. elongation step was extended to5 minutes. The PCR products were detected using SYBR-green I. EPO-R wasdetected in cardiac samples of normal rat heart tissue and in tissuesubjected in vitro to a 30 minute ischemic period irrespective oftreatment with EPO.

To confirm specificity of the product, the amplified fragments weretreated for 3 hours with the restriction enzyme NciI for partialdigestion and separated on 2.5% agarose gels by gel-electrophoresis andstained with ethidium bromide. Restriction analysis confirmed splicingof the obtained product in two fragments of the expected size (34 and 39bp, FIG. 1).

In contrast to EPO-R, we were unable to detect EPO mRNA in rat heartusing the real-time RT-PCR method described by Neumcke et al. (1999)(while brain tissue was positive in the same PCR reaction).

Western Blotting

Western blotting was performed according to standard methods (Brundel etal., 1999) on midpapillary slices from the left ventricle of rat heart,which were snap frozen in liquid nitrogen. In brief, frozen LV tissues(˜50 mg) were homogenized in 1 ml of ice-cold protein lysis buffer andprotease inhibitors. The homogenates were then centrifuged for 20minutes at 4° C. at 14,000 rpm, and the supernatant was transferred intoa clean tube and kept on ice. Protein concentration was determined byusing a standard protein assay (Bio-Rad protein assay, Bio-Rad,Richmond, Calif.). Protein samples (50 μg) were subjected to SDS-PAGE on7.5% acrylamide gels, and then transferred to PVDF membranes using a wettransfer unit (for 3 hours at 100 mA). The membranes were then blockedfor 20 minutes with Tris-buffered saline containing 0.04% Tween 20 plus5% non-fat dried milk, after which they were incubated for 3 hours withthe primary antibody in Tris-buffered saline containing 0.04% Tween 20;1:100 dilutions for the rabbit polyclonal anti-EPO-R antibody (C20,Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-EPO antibody (H-162,Santa Cruz Biotechnology, Santa Cruz, Calif.), and 1:1000 dilutions formouse monoclonal anti-phosphorylated ERK1/ERK2 antibody (#9106S, NewEngland Biolabs, Beverly Mass.). Blots were incubated for 1 hour withHRP-conjugated secondary antibody prior to the development using an ECLkit (Amersham). Our results demonstrate that both EPO and theEPO-Receptor (EPO-R) are expressed on the protein level in Langendorffperfused hearts (FIG. 2). Expression levels of both EPO and EPO-R appearunaffected by ischemia reperfusion and by the application of EPO. In thenext experiment, rat hearts were in vivo exposed to 10 U/ml EPO for 20minutes. With the use of Western blotting, we found an increase in aphosphorylated MAPK, notably ERK1 and, to a lesser extent, in ERK2 (FIG.2).

In summary, the Western blot demonstrates the presence of EPO and itsreceptor in cardiac tissue. We found EPO-R mRNA in cardiac tissue, butwere unable to detect EPO mRNA, suggesting that EPO is not locallyproduced.

Finally, we found EPO to change levels of phosphorylated MAPK,especially pERK-1, thus implying a functional role of EPO-R in cardiactissue. This may have important implications for the application of EPOin heart failure, as the extracellular signal-regulated kinases pathway(ERK1/2) has been recognized as an important regulator of cardiachypertrophy and myocyte survival in response to hypertrophic agonistsand stress stimuli (Bueno and Molkentin, 2002).

Immunohistochemistry

To evaluate the EPO and EPO-R expression pattern in rat heart tissue,complete mid-ventricular myocardial slices were obtained from thecontrol rat group. Tissue sections were fixed and paraffin-embedded.Histological slices of approximately 3 μm were sectioned, dewaxed andrehydrated with graded ethanol. The sections were incubated withanti-EPO-R antibody (C20, Santa Cruz Biotechnology, Santa Cruz, Calif.)and with anti-EPO antibody (H-162, Santa Cruz Biotechnology, Santa Cruz,Calif.) using experimental methods well known to persons skilled in theart of immunohistochemistry. A two-step indirect peroxidase detectionsystem was employed to visualize the expression pattern of EPO andEPO-R. All incubations were performed at room temperature and negativecontrols omitting the primary antibody were performed simultaneously.Using these immunohistochemistry in non-ischemic rat heart tissue, EPOexpression was found in a number of rats (n=4), where the EPO expressionappeared to be limited to arterioles and capillaries. No EPO expressionwas found in cardiomyocytes or in fibrocytes. The expression of EPO-Rwas also mostly restricted to arterioles and capillaries, although thecardiomyocytes showed a weak staining for EPO-R.

These findings further emphasize a possible role of EPO and EPO-R inangiogenesis.

Human Heart

Sections of formaline-fixed paraffin embedded human heart are obtainedfrom routine autopsy cases (Dept. Pathology, Academic HospitalGroningen). Normal autopsy material harboring no cardiac pathology isobtained from at least 10 individuals. This material is used for Westernblotting and immunohistochemistry as described above for the rat hearttissue.

Example 2 Effect of EPO in Acute Ischemic Events

The EPO-receptor (EPO-R) is found to be expressed at high concentrationsin neuronal tissues (Digicaylioglu et al. 1995; Juul et al. 1997). Theeffects caused by (temporary) hypoxia due to cerebral ischemia may bemitigated by administering erythropoietin (EPO), as disclosed in WO00/35475. Digicaylioglu and Liptyon (2001) have shown thatpreconditioning with EPO protects neurons in ischemic injury models andprevents apoptosis. As disclosed herein, EPO and the EPO-R are alsoexpressed in cardiac tissue. Cardiac tissue that is susceptible tohypoxia may, therefore, benefit from treatment with EPO (see also e.g.WO 00/61164, WO 01/82952).

Apoptosis and the release of purines from the heart are measured todetermine the effect of EPO in circumstances in which the heart tissuebecomes ischemic. For this, ischemic/reperfusion (I/R) experiments inisolated rat hearts suspended in a so-called Langendorff apparatus (VanGilst et al. 1988) are performed with and without the administration ofEPO, using methods generally known to persons skilled in the art. Therecombinant EPO is preferably obtained as described in WO 00/63403 usingpurification methods known to persons skilled in the art of proteinproduction and isolation (see also PCT/NL02/00686). An alternativesource of EPO is the commercially available epoetin alpha (EPREX®). Fourseparate experimental groups are used, each comprising eight SpragueDawley (SD) rats. Each rat weighs approximately 250 grams. These groupsare:

-   -   SD rats without I/R, without EPO    -   SD rats without I/R, with EPO    -   SD rats with I/R, without EPO    -   SD rats with I/R, with EPO

The rats are anesthetized and the heart is rapidly excised. The aorta isimmediately retrogradely perfused. Coronary flow (CF) is measured by amicroprocessor, which controls the perfusion pressure by adjusting theperistaltic perfusion pump. CF, heart rate (HR), and left ventricularpeak pressure are monitored continuously and stored in a computerdatabase. After equilibrating for 15 minutes, baseline parameters aremeasured. Ischemia is induced by ligation of the left coronary arteryfor 15 minutes. Then, reperfusion is induced by releasing the ligatureand the hearts are allowed to recover for 15 minutes.

Purine release from the heart has been shown to reflect myocardialdamage (Van Jaarsveld et al. 1989). The coronary effluent dripping fromthe heart is collected for measurement of purines released by themyocardium. Baseline samples are collected after stabilization of thepreparation, and coronary effluent is sampled after 15 minutes ischemiaand after 15 minutes of reperfusion, and purines are measured byhigh-liquid performance chromatography (HPLC). The general trend is thatinitial purine values released from the coronary effluent fromnon-EPO-treated animals start off at higher values, while the decreaseof purine over time appears to be slower, as compared to EPO-treatedanimals.

At the end of the experiments, hearts are weighed and a midpapillaryslice from the left ventricle is cut out and fixed. The non-infarctedpart of the heart (posterior wall, IV septum) is snap-frozen in liquidnitrogen. As described above, polyclonal antibodies against EPO andEPO-R are applied to determine the expression of both proteins.

Apoptosis is detected as follows. Sections from paraffin-embedded tissueblocks are placed on coated slides for in situ detection of apoptoticcells. Nuclear DNA fragments are visualized by an enzymatic reaction,using the ApopTag in situ apoptosis detection kit (Oncor, GaithersburgUSA) following the manufacturer's instructions. Number and distributionof stained cells, morphologic nuclear features and intensity of stainingare evaluated.

Example 3 Effect of EPO in Chronic Ischemia Model Systems

Myocardial infarction is induced in rats and the role of EPO, which isadministered in vivo, is determined by measuring Left VentricularPressure (LVP), infarct size, apoptosis and microvascular density. Forthis, SD rats are either sham-operated (SH) or myocardial infarcted (MI)and treated with EPO (see above) in a concentration of 400 units per kgsc, or with saline, every day for four weeks. Four separate experimentalgroups are used, each comprising eight SD rats. Each net weighsapproximately 250 grams. The groups used are:

-   -   SD rats with sham operation, without EPO    -   SD rats with sham operation, with EPO    -   SD rats with myocardial infarction, without EPO    -   SD rats with myocardial infarction, with EPO

The myocardial infarction model has been described elsewhere (Pinto etal. 1993). In brief, anesthesia is induced and a left-sided thoracotomyis performed and MI is created by ligating the left coronary artery witha 6-0 silk suture, 1-2 mm after the bifurcation with the aorta. Insham-operated rats, the same operation will be executed, withoutligating the suture.

The Left Ventricular (LV) function is determined as follows. After fourweeks, rats are anesthetized and the right carotid artery is cannulatedwith a pressure transducer catheter. After a 3 minute period ofstabilization, maximal LVP, LV end-diastolic pressure (LVEDP) and heartrate are recorded. Hereafter, the catheter is withdrawn to measuresystolic blood pressure in the aortic root. As indices of globalcontractility and relaxation, the maximal rates of increase and decreasein LVP (systolic dP/dt and diastolic dP/dt) is determined, which will befurther corrected for peak systolic LVP.

The infarct size is determined by histological analysis by staining forLDH using general methods known to persons skilled in the art. Totalepicardial and endocardial circumference of the left ventricle andepicardial and endocardial scar length of the infracted area aredetermined by means of a computerized planimeter. Infarct size iscalculated by dividing the sum of the scar lengths by the sum of thetotal circumferences, as previously described in detail (Pinto et al.1993). Furthermore, apoptosis is measured as described above.

The microvascular density is determined as follows (Loot et al., 2002).The paraffin-embedded LV slice is cut and stained with hematoxylin-eosinfor histological analysis to calculate infarct size and with RECA-1antibody to visualize microvessels using methods known to personsskilled in the art. Microvessel density per mm² is measured in thespared myocardium (opposing the infarction, usually ventricular septumor posterior wall). From each rat, seven to ten microscopic high-powerfields with transversely sectioned myocytes are digitally recorded withappropriate software. The microcirculation is defined as vessels beyondthe third order arterioles, with a diameter of 150 μm or less, supplyingtissue between arterioles and venules. Myocyte surface areas aremeasured by morphometry, selecting myocytes with a central nucleus withthe largest possible surface area with image analysis software (Loot etal., 2002).

Example 4 Determination of EPO and EPO-R Levels in Chronic Ischemia inHuman Heart

The expression levels of EPO and EPO-R are determined by the level ofmRNA, using a semi-quantitative Reverse Transcriptase Polymerase ChainReaction (RT-PCR) technique. For this, total RNA is isolated using theacid guanidium thiocyanate lysis method (Chomczynski and Sacchi 1987).The RNA is obtained from tissue from patients with ischemic heartfailure. The tissue is removed during cardiac catheterization by rightventricular endomyocardial biopsy from the right jugular or femoralvein, using standard techniques known to persons skilled in the art.Reverse transcription of RNA and amplification of cDNA is performed byRT-PCR. The cDNA of interest and the cDNA of the housekeeping enzymeGAPDH are detected by real-time RT-PCR as described above.

REFERENCES

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1. A method of treating a patient suffering from chronic heart failure,the method comprising: producing erythropoietin (EPO) in an isolatedhost cell expressing at least the Early Region 1A (E1A) protein of anadenovirus, wherein said host cell is a cell as deposited under EuropeanCollection of Animal Cell Cultures (ECACC) no. 96022940; preparing amedicament comprising said EPO for the treatment of a patient sufferingfrom chronic heart failure; and administering said medicament to saidpatient.
 2. The method according to claim 1, wherein said patient isnon-anemic.
 3. The method according to claim 1, wherein the subject isnon-anemic.
 4. A method of treating a subject suffering from chronicheart failure, the method comprising: administering to the subjecterythropoietin (EPO), wherein the EPO has been produced by a processcomprising: expressing EPO in an isolated host cell that expresses EarlyRegion 1A (E1A) protein of an adenovirus, wherein said host cell is acell as deposited under European Collection of Animal Cell Cultures(ECACC) no. 96022940.